Folding and Interaction of Subunits at the Antibody Combining Site

The Fv fragment derived from mouse myeloma protein 315, possessing anti-dinitrophenyl (DNP) activity, is composed of two subunits, the peptide chains V_L and V_H. In 8 M urea there is a complete dissociation of V_L and V_H and an approximately twofold increase in the fluorescence emission of Fv with a characteristic red shift of 11 nm. Upon dilution of Fv from 8 M urea into neutral buffer full regain of activity was observed, concomitant with regain of native fluorescence spectrum. The decrease in fluorescence upon dilution from 8 M urea was used to follow the renaturation process of Fv. At relatively high protein concentration (2.5 × 10^-6M) two steps were observed during renaturation: a fast one, which is completed in less than 30 s, and a slower step, which proceeds for approximately 20 min. The fast process represents the refolding and association of V_L and V_H to form an active Fv, whereas the slow step is attributed to the formation of “incorrect” associates between V_L and V_H which slowly reshuffle to the thermodynamically stable active Fv. Indeed, at low protein concentration (1.5 × 10^-8 M) only the fast step is observed and renaturation is completed in less than 30 s. The presence of hapten does not affect the rate of renaturation of Fv. Reoxidation of Fv completely reduced in 8 M urea was also found to yield a fully active Fv. Since either V_L or V_H have only one intrachain disulfide bond, reoxidation was performed at high protein concentration (3 mg/ml) in 8 M urea followed by dilution into neutral buffer. This demonstrates that variable domains not only exist in immunoglobulin structure but can also fold correctly independent of the rest of the peptide chains.